To understand how genes are differentially regulated in response to environmental and physiological changes, one must elucidate the underlying regulatory mechanisms. The nitrate assimilation pathway provides a versatile system for studying such mechanisms. Nitrate assimilation involves the uptake and then reduction of nitrate to ammonia by the enzymes nitrate reductase (NR) and nitrite reductase (NiR). The transport systems and reductases in this pathway and the genes that encode them are tightly controlled, responding to key environmental, metabolic and hormonal signals. In addition, genes involved in organic acid metabolism, amino acid synthesis and ferredoxin reduction are coordinately regulated with those in nitrate assimilation. Our long range goal is to elucidate the regulatory mechanisms that govern nitrate assimilation and associated pathways. To pursue this goal, studies of the regulation and function of the NRT1.1 (CHL1) transporter and NIA1 (NR) gene in the plant Arabidopsis thaliana will be performed. CHL1 is an auxin and wound-induced gene that is targeted to and functions in nascent shoot organs, root tips and guard cells; and it contains an auxin enhancer and a potential auxin response element. NIA1 is regulated by nitrate, light and sucrose and contains a nitrate enhancer. Our specific aims are to (1) elucidate the mechanisms of auxin regulation of CHL1 by mutant and promoter analyses, (2) characterize the wounding-induced response of CHL1 by promoter, mutant and microarray analyses, (3) investigate CHL1 function in and targeting to stomates, (4) examine nitrate regulation of NIA1 by promoter and mutant analysis, (5) isolate and characterize regulatory mutants by chlorate selections and targeted insertional mutagenesis, and (6) define nitrate-induced transcriptional responses in the absence of nitrate reduction and in root tips by microarray analysis. These studies will provide insights into basic biological mechanisms of gene regulation and environmental responses. This work will also contribute to our understanding of nitrate metabolism, which has helped in detecting and eliminating nitrate from water supplies where it can have detrimental health effects.